Repeated intermittent exposure to amphetamine leads to long-lasting sensitization of its psychomotor stimulant effects, such that re-exposure to the drug weeks to months later produces enhanced locomotor responding and self-administration of the drug. These findings support the proposal that sensitization of the appetitive effects of amphetamine may underlie the transition from casual drug use to drug craving and abuse. The induction of this drug-induced sensitization necessarily involves exposure to the drug in association with a complex of environmental stimuli. Repeated pairings of drugs with environmental stimuli leads to the formation of associations between the two such that these stimuli come to elicit behavioral and biochemical responses similar to those produced by the drug itself. Thus, it can be easy to confound the separate contributions of associative conditioning and non-associative sensitization to the enhanced responding observed following repeated drug exposure. One method for distinguishing between these two processes is to compare the effects of systemic amphetamine exposure to those of amphetamine applied directly to the ventral tegmental area (VTA). Amphetamine is known to act in the VTA to initiate sensitization, but fails to produce conditioning when applied to this site. Thus, by comparing the effects of VTA and systemically administered amphetamine, the associative and non-associative consequences of repeated exposure to the drug can be studied in relative isolation. The experiments in this grant proposal aim to better characterize both the neurochemical and morphological underpinnings of associative conditioning and non-associative sensitization, and will test two main hypotheses. Hypothesis 1: The activity of specific proteins in the nucleus accumbens (NAcc) during drug-conditioning are necessary for the formation of learned associations linking the drug environment to stimuli. Preliminary findings demonstrate, and the proposed experiments will further explore, how inhibition of the protein cyclin-dependent kinase 5 (cdk5) in the NAcc during drug exposure blocks associative conditioning while preserving non-associative sensitization. Furthermore, by comparing other protein changes in the NAcc caused by repeated systemic or VTA amphetamine exposure, we will be able to identify other proteins correlated either with conditioning or sensitization. Hypothesis 2: Specific populations of neurons undergo conditioning-related neuroanatomical plasticity. Using c-Fos as a marker to identify NAcc cells contributing to associative conditioning, we will inject these neurons with the carbocyanine neuronal tracer DiI in order to characterize their dendritic spine morphology using confocal 2-photon microscopy. PUBLIC HEALTH RELEVANCE: Drug addiction is a disease that is uniquely costly to society and affects the health, productivity, and well-being of individuals in various age groups and demographics. Much published research demonstrates how prolonged drug use produces changes in the brain. The proposed experiments extend this approach to study how these changes are regulated at the neuronal level by environmental factors that may encourage drug-seeking. Understanding how drug-paired environments regulate brain processes underlying compulsive drug use may lead to novel therapeutic and pharmacological treatments for the disease.